Cell-permeable imaging sensors and uses thereof

ABSTRACT

The disclosure relates in some aspects to imaging agents, and in particular, imaging agents for sensing of calcium signaling. According to some embodiments of the disclosure, contrast agents for magnetic resonance imaging and related technologies are provided, and methods of making and using the contrast agents.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S.Provisional Application No. 62/597,875, filed Dec. 12, 2017 and entitled“CELL-PERMEABLE IMAGING SENSORS AND USES THEREOF,” which is incorporatedherein by reference in its entirety for all purposes.

GOVERNMENT FUNDING

This invention was made with Government support under Grant Nos. R01DA038642 and U01 NS090451 awarded by the National Institutes of Health.The Government has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates in some aspects to imaging agents.

BACKGROUND OF THE INVENTION

Available imaging techniques for measuring large-scale signalingdynamics in intact organisms have been limited. With optical techniquesit is possible to perform functional imaging of signaling dynamics atdepths of up to about two millimeters in tissue, but for most vertebratespecies this only represents a small fraction of the volumes ofexperimental interest. Implantable endoscopes and prisms permitmeasurements in deeper structures, but only over limited fields of view.Hybrid techniques like photoacoustic tomography achieve submillimeterimaging resolution with considerably greater tissue penetration thanconventional optics. Thus, imaging techniques are often limited by sharptrade-offs between depth and resolution.

SUMMARY OF THE INVENTION

Aspects of the disclosure relate to sensors and related methods forimaging. In some embodiments, cell-permeable imaging sensors areprovided that facilitate imaging of cellular ion signaling (e.g.,calcium signaling). In some embodiments, magnetic resonance imaging(MRI) provides a powerful technique for evaluating ion signaling (e.g.,calcium ion) in animals and humans (e.g., in wide-field, deep-tissuecontext) using the sensors provided herein. In some embodiments, MRIachieves a combination of unlimited depth penetration, relatively high3D spatial resolution (<100 μm in some contexts), and sensitivity to awide variety of contrast mechanisms. Accordingly, in some aspects, novelmagnetic resonance imaging (MRI) sensors are disclosed herein. In someembodiments, these sensors are specific for detecting ions in cells,such as calcium ions. In some embodiments, the sensors provided hereinmay be applied to noninvasive neuroimaging, muscular (including cardiac)imaging, immunological activation, or imaging of secretory cells inother organs of animals and humans. Furthermore, in some embodiments,sensors provided herein are powerful tools for functional MRI (MRI) andhave various applications, including but not limited to: functionalvisualization of brain activity in healthy and disease/disordered modelsat preclinical studies, neuroimaging relevant to surgical planning,peripheral neuroimaging relevant to significant neuropathies, tests ofcardiac or skeletal muscle function, and monitoring lymphatic functionin inflammation and cancer.

In some aspects, an ion sensor (e.g., calcium sensor) reported hereincomprises a small-molecule complex comprising a transition metal (e.g.,manganese). In some embodiments, the small-molecule complex comprises alipophilic, branched chelating moiety complexed with a transition metal.In some embodiments, the lipophilic, branched chelating moiety is linkedto an analyte binding moiety (e.g., a cell-permeable calcium chelatorsuch as BAPTA) (See, e.g., FIG. 5). In some embodiments, the sensorcomprises ManICS1 (see, e.g., FIG. 2, 16). In some embodiments, thesensor comprises the structure in FIG. 2, 13. In some embodiments, thesensor comprises ManICS1-AM (see, e.g., FIG. 2, 14). In someembodiments, micromolar concentrations of ManICS1-AM cause enhancementof T1-weighted MRI signal. In some embodiments, ManICS1-AM accumulatesand is retained cytosolically in cells and upon enzymatic reaction turnsinto its active form ManICS1. In some embodiments, cells loaded withManICS1-AM undergo stimulus-induced changes in T1-weighted MRI contrastthat parallel responses measured optically using a fluorescent calciumindicator.

In some aspects, a sensor comprising the structure, Y-L-Z is provided.In some embodiments, Y is an analyte binding moiety (e.g., that bindscalcium ions). In some embodiments, Z is a lipophilic, branchedchelating moiety. In some embodiments, L is a linker that covalentlylinks Y and Z.

It should be appreciated that the foregoing concepts, and additionalconcepts discussed below, may be arranged in any suitable combination,as the present disclosure is not limited in this respect. Further, otheradvantages and novel features of the present disclosure will becomeapparent from the following detailed description of various non-limitingembodiments when considered in conjunction with the accompanyingfigures.

In cases where the present specification and a document incorporated byreference include conflicting and/or inconsistent disclosure, thepresent specification shall control. If two or more documentsincorporated by reference include conflicting and/or inconsistentdisclosure with respect to each other, then the document having thelater effective date shall control.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures may be represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIGS. 1A to 1D depict cell permeable sensors for calcium-dependentmolecular fMRI. FIG. 1A shows a cell permeable paramagnetic platform(complex, molecule portion below —R—, Mn-PDA candidate complexes shownat top right), a BAPTA-based calcium chelator (molecule portion above—R—), and a linker connecting them (—R—). Prior to cell entry (top) theBAPTA carboxylates were protected with cleavable acetoxymethyl (AM)esters, and water (H₂O) exchange was hypothesized to take place at theparamagnetic metal center (circle at center of paramagnetic platform),leading to T₁-weighted MRI signal enhancement (labeled MRI). When theagent entered cells (bottom left), the AM esters were cleaved,liberating the sensor in its calcium-free “off” state; in this state,water exchange may be blocked by interactions between the BAPTA moietyand Mn³⁺, leading to low MRI signal. When calcium bound to the sensor(bottom right), the MRI signal increased again as interactions betweenthe BAPTA and paramagnetic platform were reduced. FIG. 1B depictsresults of an evaluation of interactions between BAPTA and Mn-PDAcontrast agents. In the absence of BAPTA, the Mn³⁺ complexes did notproduce calcium-dependent T₁-weighted MRI contrast changes (top), but inthe presence of 1.1 eq BAPTA, both MnL1 and MnL3 displayed sensitivityto the addition of calcium (bottom). FIG. 1C depicts longitudinalrelaxivity (r₁) values corresponding to the conditions in FIG. 1B. FIG.1D depicts optical spectra of contrast agents MnL1 and MnL3 in thepresence of BAPTA after incubation for 0 h (solid) or 24 h (dashed),indicating comparative stability of MnL3.

FIG. 2 depicts a synthesis of ManICS1 and ManICS1-AM [The followingconditions are noted: vi) NaOH (96%); vii) 7, AcOCH₂Br,N,N-diisopropylethylamine (DIEA) (67%); viii) NaH₂PO₄, NaClO₂ (95%); ix)9, (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate)(PyBOP), DIEA, NH₂—CH₂(CH₂OCH₂)₄ CH₂—NH(tert-butyloxycarbonyl)(NH₂-PEG₄-NHBoc) (55%); x) trifluoroacetic acid (TFA), dichloromethane(DCM) (98%); xi) L₃COOH (12 without Mn), PyBOP, DIEA (54%); xii)manganese (III) acetate Mn(OAc)₃.2(H₂O) (85%); xiii) 10% (v/v) celllysate, (3-(N-morpholino)propanesulfonic acid) (MOPS) (25 mM, pH 8);xiv) KOH (6 eq), H₂O (89%); xv) MnL₃COOH, PyBOP, DIEA (38%)].

FIGS. 3A to 3C show that ManICS1 reported calcium-dependent MRI signalchanges in buffer, and that MnICS1-AM underwent enzymatic hydrolysis.FIG. 3A shows T₁-weighted images of ManICS1 with addition of varyingmicromolar concentrations of CaCl₂ (top) or MgCl₂ (bottom) in MOPSbuffer, pH 7.4. FIG. 3B shows relaxivity changes for Calcium (top curve)and Magnesium (bottom curve) corresponding to conditions in FIG. 3A.Ligand-depleting bimolecular binding models were used to generate thefitted curves shown. FIG. 3C shows reversed phase HPLC traces of 10%(v/v) HEK293 cell lysate (Lysate), ManICS1(+ManICS1), ManICS1-AM,ManICS1-AM treated with cell lysate for 5 h (+Lysate (5 h) directlybelow ManICS1-AM), ManICS1-Et, and ManICS1-Et treated with cell lysatefor 5 h (+Lysate (5 h) directly below ManICS1-Et). Vertical dashed lineindicates expected elution time of ManICS1.

FIGS. 4A to 4D show that ManICS1 reported calcium-dependent MRI signalchanges in cells. FIG. 4A shows a washout time course of ΔR₁/R₁ vs. timefor HEK293 cells preincubated with ManICS1-AM (top) or ManICS1 acid form(bottom). ManICS1-AM was selectively retained, e.g., which may occur dueto intracellular cleavage of its AM esters. FIG. 4B showscompartment-specific accumulation of manganese in cells incubated withManICS1-AM and ManICS1, showing enhanced labeling of both cytosolic(left of each set of three) and membranous (right of each set of three)fractions, as indicated by inductively coupled plasma optical emissionspectrometry (ICP-OES) of fractionated labeled cells. FIG. 4C showscalcium responses measured in HEK293 cells labeled with ManICS1-AM (leftplot) or Fura-2-AM (right plot) and treated with cell stimulantsthapsigargin (Th), carbachol (Ch), the Ca²⁺ ionophore calcimycin (Ca),or arachidonic acid (AA). MRI responses diagrammed at left paralleledfluorescence responses measured under equivalent conditions at right.FIG. 4D shows titration of extracellular calcium concentration in thepresence of calcimycin in ManICS1-AM-loaded cells. A midpoint ofcalcium-induced changes occurred at [Ca²⁺]=5 micromolar (μM). Insetcompares MRI scans of cell pellets imaged in the absence (left) vs.presence (right) of calcium.

FIG. 5 shows molecular structures of intracellular calcium sensors forMRI. AM is acetoxymethyl.

FIG. 6 depicts a synthesis of ManICS1 and ManICS1-AM.

FIGS. 7A to 7F show that ManICS1 reports calcium-dependent MRI signalchanges in cells. FIG. 7A shows the washout time course of ΔR₁/R₁ vs.time for HEK293 cells preincubated with 10 μM ManICS1-AM or ManICS1 acidform. Elemental analysis of cytosolic fractions from incubated celllysates (inset) indicates intracellular manganese accumulation inManICS1-AM (M1AM)-treated cells but not ManICS1 (M1)-treated cells. FIG.7B shows titration of extracellular calcium concentration equilibratedin the presence of 10 μM of the Ca²⁺ ionophore calcimycin inManICS1-AM-loaded cells. A midpoint of calcium-induced changes occurs at[Ca²⁺]=5 μM. Inset compares MRI scans of cell pellets imaged in theabsence (left) vs. presence (right) of 1 mM calcium. FIG. 7C showscalcium responses measured from HEK293 cells labeled with ManICS1-AM andtreated by extracellular addition of chemical stimulants (left).Significant R₁ changes were observed in response to calcimycin (Ca) andarachidonic acid (AA) (p≤0.001), but not thapsigargin (Th) or carbachol(Ch) (p≥0.2). FIG. 7D shows responses measured by fluorescencespectroscopy from cells loaded with the fluorescent calcium indicatorFura-2FF-AM under stimulation conditions as in FIG. 7C. FIG. 7E showscells were loaded by incubation with 40 μM ManICS1-AM, transfected withthe light sensitive Orai calcium channel activator BACCS2, andstimulated with 480 nm light (diagram at left). The top time courseshows resulting changes in the T₁-weighted signal as a function of timebefore, during, and after stimulation (vertical gray bar). Inset at topdepicts image snapshots binned over successive 240 s windows during thetime series portion indicated by dashed lines and indicating percentsignal changes observed at a voxel level. Stimulus-dependent signalchanges were not observed in analogous experiments performed using cellslacking BACCS2 (middle) or cells labeled with MnL3 instead of ManICS1-AM(bottom). FIG. 7F shows fluorescence time course observed fromBACCS2-expressing cells incubated with 5 μM X-Rhod-1-AM and stimulatedas in FIG. 7E.

FIGS. 8A to 8F show ManICS1-AM enables detection of neural activation inrat brain. FIG. 8A shows T₁-weighted MRI showing broad contrastenhancement following infusion of 15 μL ManICS-1 (left) orcalcium-insensitive control agent (MnL1F, right) into rat striatum.Rostrocaudal coordinates with respect to bregma indicated. FIG. 8B showsestimated volume of signal enhancements within 10% of peak values afterManICS1-AM infusion in four animals (mean=36±7 μL). In FIG. 8C, a plotof peak MRI signal in ManICS1-AM infused brain regions over time shows amean signal enhancement of 20±2% with an average of 0.9% signal decreaseper hour with respect to image intensity in unenhanced tissue (bold);the signal decrease from 30 to 90 minutes post-infusion was notstatistically significant (t-test p=0.42, n=6). Data from individualanimals shown in gray. FIG. 8D shows 1 μL K⁺ infusion causes T₁-weightedMRI signal increases in the presence of predelivered ManICS1-AM (left)but not MnL1F (right). Average peak signal change across multipleanimals (n=5) is indicated by the color scale superimposed on a highresolution T₁-weighted image of a representative rat. Scale bar=3 mm.FIG. 8E shows the region of interest analysis, which shows the timecourse of signal changes observed during K⁺ or Na⁺ in the presence ofManICS1 (top and middle, respectively), and during K⁺ stimulation in thepresence of calcium-insensitive MnL1F (bottom). FIG. 8F shows MRI signalchanges observed in individual animals within one minute of K⁺ or Na⁺treatment offset for the conditions in FIG. 8E. Mean signal changesobserved during potassium stimulation in the presence of ManICS1-AM wassignificantly greater than results from both controls (t-test p≤0.016,n=5).

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

Certain ions (e.g., calcium, potassium, sodium) are important for signaltransduction in cells, where they help coordinate biological processesranging from embryonic development to neural function in the brain.Accordingly, in some embodiments, sensors and related methods describedherein may be used to detect such ions and other analytes to gain afunctional and/or physiological understanding of the biology of majordisease areas, e.g., in neuroscience, which may lead to the discovery ofaddressable functional mechanisms in health and disease.

In some embodiments, sensors and related methods provided hereinfacilitate drug development by providing new tools for pharmacologicalscreening and characterization. In some embodiments, using the ionresponse (e.g., calcium signaling as a readout facilitates the study ofdrug or physiological effects.

In some embodiments, sensors and related methods provided herein may beused for clinical diagnostic imaging. In some embodiments, sensors maybe used, for example, to visualize cell signaling for diagnosis innumerous conditions, including neurological and muscular disorders, aswell as immunological and endocrine conditions and others.

Sensors

In some embodiments, sensors provided herein comprise the structureY-L-Z, wherein: Y is an analyte binding moiety; Z is a lipophilic,branched chelating moiety; and L is a linker that covalently links Y andZ. Accordingly, in some embodiments, the sensor comprises an organic oran organometallic compound. In some embodiments, the sensor furthercomprises an additional Y, Z, L, -L-Y, or -L-Z covalently bound to Y orZ. In some embodiments, the sensor further comprises an additional L-Zcovalently bound to Y (e.g., Z-L-Y-L-Z). In some embodiments, the sensorfurther comprises an additional -L-Y covalently bound to Z (e.g.,Y-L-Z-L-Y).

Various analytes that may be detected using the sensors described hereininclude metal ions, nitric oxide, or other species associated withnormal biological function. The analyte may be present within a subject(e.g., a human), and, in some cases, may be present within a cell. Insome cases, the analyte may be present within a subject in a locationexterior to a cell. In some cases, the analyte is a metal ion, such as atoxic metal ion. Examples of metal ions include, but are not limited to,Zn²⁺, Ca²⁺, Hg²⁺, Cd²⁺, Pb²⁺, Na⁺, Mg²⁺, Mn²⁺, Fe²⁺, Co²⁺, Ni²⁺, andCu²⁺. In some cases, the analyte is Zn²⁺, Ca²⁺, Mg²⁺, Hg²⁺, Cd²⁺, orPb²⁺. In some embodiments, the analyte may be a zinc ion or calcium ion.In certain embodiments, the analyte may be a magnesium ion.

In some embodiments, the analyte may be a calcium ion. Calcium is acomponent of cellular signaling systems and participates in brainfunction among other processes. Muscles (including the heart) may alsodepend on regulation of calcium, and calcium has been implicated inmyocardial dysfunction. In one embodiment, a sensor may be useful in thedetermination of calcium ions within a cell, such as a muscle cell orneuronal cell.

In some embodiments, the analyte binding moeity contains at least onearomatic group. In some embodiments, contains at least one aromaticgroup selected from the group consisting of monocyclic aryl (e.g.,phenyl), bicyclic aryl (e.g., naphthyl), monocyclic heteroaryl (e.g.,pyridyl, pyrimidyl, pyrrolyl, imidazolyl), and bicyclic heteroaryl(e.g., quinolyl, indolyl).

In certain embodiments, the analyte binding moiety is apolyaminocarboxylate chelator. In certain embodiments, the analytebinding moiety is a polyaminocarboxylate chelator selected from thegroup consisting of polyaminocarboxylate chelator is selected from thegroup consisting of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether N,N,N′,N′-tetraacetic acid (EGTA),diethylenetriaminepentaacetic acid (DTPA),2-[(2-amino-5-methylphenoxy)methyl]-6-methoxy-8-aminoquinoline-N,N,N′,N′-tetraaceticacid (quin-2),1-(2-nitro-4,5-dimethoxyphenyl)-1,2-diaminoethane-N,N,N′,N′-tetraaceticacid (DM-nitrophen), and o-aminophenol-N,N,O-triacetic acid (APTRA). Insome embodiments, the analyte binding moiety selectively chelates metalions. In some embodiments, the analyte binding moiety isethylenediaminetetraacetic acid (EDTA). In certain embodiments, theanalyte binding moiety is ethylene glycol-bis(β-aminoethylether)N,N,N′,N′-tetraacetic acid (EGTA). In certain embodiments, theanalyte binding moiety is a metal ion-selective crown ether.

In some embodiments, an analyte binding moiety comprises a selectivemetal ion chelating moiety. In some embodiments, the selective metal ionchelating moiety is cell permeable. In some embodiments, the selectivemetal ion chelating moiety comprises a selective calcium chelatingmoiety derived from 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraaceticacid (BAPTA). In some embodiments, BAPTA, which is highly polar andmembrane-impermeant in its Ca²⁺-binding acidic form, may be modifiedwith modifying groups (e.g., with acetomethoxyl (AM) ester groups) toproduce a neutral, inactive complex that is readily internalized insidecells. In certain embodiments, the selective calcium chelating moietyderived from BAPTA does not comprise carboxyl moieties. In certainembodiments, in the selective calcium chelating moiety derived fromBAPTA, each of the moieties —C(═O)OH of BAPTA is replaced with themoiety —C(═O)OR, wherein each instance of R is independently substitutedor unsubstituted C₁₋₆ alkyl. In certain embodiments, each instance of Ris C₁₋₆ alkyl or C₃₋₁₀ cycloalkyl, wherein R is unsubstituted orsubstituted with —OR¹, —C(═O)R¹, —O—C(═O)R¹, —C(═O)OR¹, —NR¹—C(═O)R¹, or—C(═O)N(R¹)₂ wherein each R¹ is independently H, substituted orunsubstituted C₁₋₆ alkyl (e.g., methyl, ethyl, propyl, butyl), orsubstituted or unsubstituted C₃₋₁₀ cycloalkyl (e.g., cyclopentyl,cyclobutyl, cyclopentyl, cyclohexyl). In certain embodiments, eachinstance of R is —CH₂—O—C(═O)—CH₃. In some embodiments, afterinternalization, the modifying groups (e.g., AM esters) undergohydrolysis (e.g., catalyzed by intracellular esterases). In someembodiments, this process may activate the sensor (also referred toherein as an indicator) and trap it in the cell cytosol, where itfunctions as a cytosolic [Ca²⁺] imaging agent.

In some embodiments, the lipophilic, branched chelating moiety iscapable of binding a metal ion, such as a paramagnetic metal ion (e.g.,Mn³⁺, Fe³⁺). In some embodiments, the analyte binding moiety is capableof binding an analyte such that an MRI and/or optical property of thesensor is shifted upon binding the analyte. As described herein, thepresence or absence of a metal ion bound to the chelating moiety(chelator group), or, the type of metal ion bound to the chelatingmoiety, may affect one or more properties of the sensor, such as an MRIor optical property. In some cases, the sensor comprises a chelatingmoiety which binds a paramagnetic metal ion, such that the sensorexhibits an MRI signal upon exposure to MRI conditions, e.g., exposureto a magnetic field. In some cases, the sensor comprises a chelatingmoiety which does not bind a metal ion or binds a diamagnetic metal ion,such that the sensor exhibits a luminescence emission or absorption uponexposure to electromagnetic radiation.

As used herein, the term “paramagnetic metal ion” refers to a metal ionhaving unpaired electrons, causing the metal ion to have a measurablemagnetic moment in the presence of an externally applied field. Examplesof suitable paramagnetic metal ions, include, but are not limited to,ions of iron, nickel, manganese, copper, gadolinium, dysprosium,europium, and the like. Mn²⁺, Mn³⁺, Fe²⁺, and Fe³⁺ are non-limitingexamples of paramagnetic metal ions. In some embodiments, a sensor orcomposition of the disclosure comprises a chelator group bound to aparamagnetic metal ion, wherein the paramagnetic ion is an ion of ironor manganese.

In certain embodiments, the lipophilic, branched chelating moietycomprises an aromatic group. In some embodiments, the lipophilic,branched chelating moiety comprises one or more cyclic moieties. In someembodiments, the lipophilic, branched chelating moiety is independentlyselected from the group consisting of optionally substituted monocycliccarbocyclyl (e.g., cyclopentyl, cyclohexyl), optionally substitutedbicyclic carbocyclyl (e.g., decahydronaphtalenyl,bicyclo[2.2.1]heptanyl), optionally substituted monocyclic heterocyclyl(e.g., tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, pyrolinyl),and optionally substituted bicyclic heterocyclyl (e.g.,decahydroquinolinyl). In some embodiments, the lipophilic, branchedchelating moiety is independently selected from the group consisting ofoptionally substituted monocyclic aryl (e.g., phenyl, anilinyl),optionally substituted bicyclic aryl (e.g., naphthyl), optionallysubstituted monocyclic heteroaryl (e.g., pyridyl, pyrimidyl, pyrrolyl,imidazolyl), and optionally substituted bicyclic heteroaryl (e.g.,quinolyl, indolyl). In certain embodiments, the lipophilic, branchedchelating moiety comprises aromatic fluorophore.

In some embodiments, the lipophilic, branched chelating moiety comprisesa planar lipophilic ligand (e.g., a planar cell-permeable aromaticfluorophore). In some embodiments, the planar lipophilic ligand belongsto a family of planar phenylenediamido (PDA)-based Mn³⁺ complexes (see,e.g., FIG. 1A, top right box) that act as T₁ MRI contrast agents and canundergo cell internalization and trapping analogous to organic AMester-derivatized fluorophores. Conjugation of such a complex to theselective metal ion chelating moiety (e.g., BAPTA-based chelator) mayresult in a candidate MRI metal ion (e.g., calcium ion) sensor withsimilar physicochemical properties to optical imaging dyes like Fluo-4(PubChem CID: 25058176), Fura-2 (PubChem. CID: 57054), and Oregon GreenBAPTA (PubChem. CID: 102028682). In some embodiments, such a probe couldbe administered to cells in modified form with modifying groups (e.g.,AM ester), which probe (also referred to herein as a sensor) would beinternalized into a cell and activated (see, e.g., FIG. 1A).

In some embodiments, interactions between the selective metal ionchelating moiety (e.g., based on BAPTA) and the planar lipophilic ligand(e.g., an Mn-PDA) of the activated sensor provide a basis fortransducing calcium concentration changes into T₁-weighted MRI signals.Accordingly, in embodiments provided herein, magnetic resonance imaging(MRI) provides for wide-field deep-tissue ion imaging (e.g., calciumimaging) in animals and humans. MRI achieves a combination of unlimiteddepth penetration, relatively high 3D spatial resolution (<100 μm insome contexts), and sensitivity to a wide variety contrast mechanisms.In some embodiments, the sensors described herein are used for real-timefunctional magnetic resonance imaging of a signaling ion (e.g., calcium,for calcium ion signaling) in cells and tissues.

In some embodiments, L, is an unbranched C₄₋₄₀ alkylene, unbranchedC₄₋₄₀ alkenylene, or unbranched C₄₋₄₀ alkynylene. In certainembodiments, 0, 1, or more methylene units of the unbranched C,₄₋₄₀alkylene, unbranched C₄₋₄₀ alkenylene, or unbranched C₄₋₄₀ alkynyleneare independently replaced with O, N, N(R^(F)), C(═O)O, C(═O)NR^(F), S,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl asvalency permits. In certain embodiments, 1 or more methylene units ofthe unbranched C₄₋₄₀ alkylene is N (e.g., —CH₂CH₂C═NCH₂—). In certainembodiments, 1 or more methylene units of the unbranched C₄₋₄₀ alkyleneis C(═O)O. In certain embodiments, 1 or more methylene units of theunbranched C₄₋₄₀ alkylene is C(═O)NR^(F). In certain embodiments, 1 ormore methylene units of the unbranched C₄₋₄₀ alkylene is optionallysubstituted carbocyclyl (e.g., cyclopentyl, cyclobutyl, cyclopentyl,cyclohexyl). In certain embodiments, 1 or more methylene units of theunbranched C₄₋₄₀ alkylene is optionally substituted heterocyclyl (e.g.,tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, pyrolinyl,dioxopyrrolidinyl) In certain embodiments, 1 or more methylene units ofthe unbranched C₄₋₄₀ alkylene is optionally substituted aryl (e.g.,phenyl

anilinyl

In certain embodiments, 1 or more methylene units of the unbranchedC₄₋₄₀ alkylene is optionally substituted heteroaryl (e.g., pyridyl,pyrimidyl, pyrrolyl, imidazolyl, tetrazolyl, triazolyl, quinolyl,indolyl). In certain embodiments, R^(F) is H. In some embodiments, R^(F)is unsubstituted C₁₋₆ alkyl (e.g., methyl, ethyl, propyl, isopropyl,butyl, pentyl, hexyl). In certain embodiments, a methylene unit isindependently substituted with oxo. In certain embodiments, a methyleneunit is independently substituted with —OR^(a) (—OCH₃ or —OCH₂CH₃). Insome embodiments, a methylene unit is substituted with halogen (e.g.,—F, —Cl, —Br, or —I. In certain embodiments, a methylene unit issubstituted with C₁₋₆ alkyl (e.g., methyl, ethyl, propyl, isopropyl,butyl, pentyl). In certain embodiments, a methylene unit is substitutedwith C₁₋₆ alkyl substituted with one or more instances of halogen (e.g.—CF₃, —CHF₂, —CH₂F, —CH₂CF₃).

In some embodiments, methods of making a sensor are provided (See, e.g.,Examples and FIG. 6).

In some embodiments, methods of using the sensor are provided. In someembodiments, the sensor is used to monitor changes in intracellularmetal ion (e.g., calcium ion) levels by MRI (e.g., proton MRI).

Chemical Definitions

The term “alkyl” refers to a radical of a straight-chain or branchedsaturated hydrocarbon group having from 1 to 20 carbon atoms (“C₁₋₂₀alkyl”). In some embodiments, an alkyl group has 1 to 12 carbon atoms(“C₁₋₁₂ alkyl”). In some embodiments, an alkyl group has 1 to 10 carbonatoms (“C₁₋₁₀ alkyl”). In some embodiments, an alkyl group has 1 to 8carbon atoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1to 7 carbon atoms (“C₁₋₇ alkyl”), in some embodiments, an alkyl grouphas 1 to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkylgroup has 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, analkyl group has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments,an alkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In someembodiments, an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). Insome embodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), propyl(C₃) (e.g., n-propyl, isopropyl), butyl (C₄) (e.g., n-butyl, tert-butyl,sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl, 3-pentanyl, amyl,neopentyl, 3-methyl-2-butanyl, tertiary amyl), and hexyl (C₆) (e.g.,n-hexyl). Additional examples of alkyl groups include n-heptyl (C₇),n-octyl (C₈), and the like. Unless otherwise specified, each instance ofan alkyl group is independently unsubstituted (an “unsubstituted alkyl”)or substituted (a “substituted alkyl”) with one or more substituents(e.g., halogen, such as F). In certain embodiments, the alkyl group isan unsubstituted C₁₋₁₂ alkyl (such as unsubstituted C₁₋₆ alkyl, e.g.,—CH₃ (Me), unsubstituted ethyl (Et), unsubstituted propyl (Pr, e.g.,unsubstituted n-propyl (n-Pr), unsubstituted isopropyl (i-Pr)),unsubstituted butyl (Bu, e.g., unsubstituted n-butyl (n-Bu),unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstituted sec-butyl(sec-Bu or s-Bu), unsubstituted isobutyl (i-Bu)). In certainembodiments, the alkyl group is a substituted C₁₋₁₂ alkyl (such assubstituted C₁₋₆ alkyl, e.g., —CH₂F, —CHF₂, —CF₃, —CH₂CHF, —CH₂CHF₂,—CH₂CF₃, or benzyl (Bn)).

The term “haloalkyl” is a substituted alkyl group, wherein one or moreof the hydrogen atoms are independently replaced by a halogen, e.g.,fluoro, bromo, chloro, or iodo. “Perhaloalkyl” is a subset of haloalkyl,and refers to an alkyl group wherein all of the hydrogen atoms areindependently replaced by a halogen, e.g., fluoro, bromo, chloro, oriodo. In some embodiments, the haloalkyl moiety has 1 to 8 carbon atoms(“C₁₋₈ haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 6carbon atoms (“C₁₋₆ haloalkyl”). In some embodiments, the haloalkylmoiety has 1 to 4 carbon atoms (“C₁₋₄ haloalkyl”). In some embodiments,the haloalkyl moiety has 1 to 3 carbon atoms (“C₁₋₃ haloalkyl”). In someembodiments, the haloalkyl moiety has 1 to 2 carbon atoms (“C₁₋₂haloalkyl”). In some embodiments, all of the haloalkyl hydrogen atomsare replaced with fluoro to provide a perfluoroalkyl group. In someembodiments, all of the haloalkyl hydrogen atoms are replaced withchloro to provide a “perchloroalkyl” group. Examples of haloalkyl groupsinclude —CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂, —CF₂Cl, and the like.

The term “heteroalkyl” refers to an alkyl group, which further includesat least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected fromoxygen, nitrogen, or sulfur within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkyl group refers toa saturated group having from 1 to 10 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC₁₋₁₀ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC₁₋₅ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC₁₋₁₀ alkyl.

The term “alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In someembodiments, an alkenyl group has 2 to 9 carbon atoms (“C₂₋₉ alkenyl”).In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C₂₋₈alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms(“C₂₋₇ alkenyl”). In some embodiments, an alkenyl group has 2 to 6carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, an alkenyl group has2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In some embodiments, an alkenylgroup has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”). In some embodiments, analkenyl group has 2 to 3 carbon atoms (“C₂₋₃ alkenyl”). In someembodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”). The oneor more carbon-carbon double bonds can be internal (such as in2-butenyl) or terminal (such as in 1-butenyl). Examples of C₂₋₄ alkenylgroups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl(C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkenyl groups as well aspentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additionalexamples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl(C₈), and the like. Unless otherwise specified, each instance of analkenyl group is independently unsubstituted (an “unsubstitutedalkenyl”) or substituted (a “substituted alkenyl”) with one or moresubstituents. In certain embodiments, the alkenyl group is anunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis a substituted C₂₋₁₀ alkenyl. In an alkenyl group, a C═C double bondfor which the stereochemistry is not specified (e.g., —CH═CHCH₃,

may be in the (E)- or (Z)-configuration.

The term “heteroalkenyl” refers to an alkenyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkenylgroup refers to a group having from 2 to 10 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenyl group has2 to 9 carbon atoms at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 8 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbonatoms, at least one double bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and for 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”). Insome embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₁₀ alkenyl.

The term “alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C₂₋₁₀alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms(“C₂₋₉ alkynyl”). In some embodiments, an alkynyl group has 2 to 8carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, an alkynyl group has2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, an alkynylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In some embodiments, analkynyl group has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”). In someembodiments, an alkynyl group has 2 to 4 carbon atoms (“C₂₋₄ alkynyl”).In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C₂₋₃alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂alkynyl”). The one or more carbon-carbon triple bonds can be internal(such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples ofC₂₋₄ alkynyl groups include ethynyl (C₂), 1-propynyl (C₃), 2-propynyl(C₃), 1-butynyl (C₄), 2-butynyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkynyl groups as well aspentynyl (C₅), hexynyl (C₆), and the like. Additional examples ofalkynyl include heptynyl (C₇) octynyl (C₈), and the like. Unlessotherwise specified, each instance of an alkynyl group is independentlyunsubstituted (an “unsubstituted alkynyl”) or substituted (a“substituted alkynyl”) with one or more substituents. In certainembodiments, the alkynyl group is an unsubstituted C₂₋₁₀ alkynyl. Incertain embodiments, the alkynyl group is a substituted C₂₋₁₀ alkynyl.

The term “heteroalkynyl” refers to an alkynyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkynylgroup refers to a group having from 2 to 10 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynyl group has2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbonatoms, at least one triple bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”).In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆alkynyl”). Unless otherwisespecified, each instance of a heteroalkynyl group is independentlyunsubstituted (an “unsubstituted heteroalkynyl.”) or substituted (a“substituted heteroalkynyl”) with one or more substituents. In certainembodiments, the heteroalkynyl group is an unsubstituted heteroC₂₋₁₀alkynyl. In certain embodiments, the heteroalkynyl group is asubstituted heteroC₂₋₁₀ alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbonatoms (“C₃₋₁₄ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C₃₋₇carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ringcarbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, a carbocyclylgroup has 4 to 6 ring carbon atoms (“C₄₋₆ carbocyclyl”). In someembodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C₅₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groupsinclude cyclopropyl (C₃), cyclopropenyl (C₃), cyclobutyl (C₄),cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅), cyclohexyl(C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and the like. ExemplaryC₃₋₈ carbocyclyl groups include the aforementioned C₃₋₆ carbocyclylgroups as well as cycloheptyl (C₇), cycloheptenyl (C₇), cycloheptadienyl(C₇), cycloheptatrienyl (C₇), cyclooctyl (C₈), cyclooctenyl (C₈),bicyclo[2.2.1]heptanyl (C₇), bicyclo[2.2.2]octanyl (C₈), and the like.Exemplary C₃₋₁₀ carbocyclyl groups include the aforementioned C₃₋₈carbocyclyl groups as well as cyclononyl (C₉), cyclononenyl (C₉),cyclodecyl (C₁₀), cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉),decahydronaphthalenyl (C₁₀), spiro[4.5]decanyl (C₁₀), and the like. Asthe foregoing examples illustrate, in certain embodiments, thecarbocyclyl group is either monocyclic (“monocyclic carbocyclyl”) orpolycyclic (e.g., containing a fused, bridged or spiro ring system suchas a bicyclic system (“bicyclic carbocyclyl”) or tricyclic system(“tricyclic carbocyclyl”)) and can be saturated or can contain one ormore carbon-carbon double or triple bonds. “Carbocyclyl” also includesring systems wherein the carbocyclyl ring, as defined above, is fusedwith one or more aryl or heteroaryl groups wherein the point ofattachment is on the carbocyclyl ring, and in such instances, the numberof carbons continue to designate the number of carbons in thecarbocyclic ring system. Unless otherwise specified, each instance of acarbocyclyl group is independently unsubstituted (an “unsubstitutedcarbocyclyl”) or substituted (a “substituted carbocyclyl”) with one ormore substituents. In certain embodiments, the carbocyclyl group is anunsubstituted C₃₋₁₄ carbocyclyl. In certain embodiments, the carbocyclylgroup is a substituted C₃₋₁₄ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 14 ring carbon atoms (“C₃₋₁₄cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl(C₅) and cyclohexyl (C₅). Examples of C₃₋₆ cycloalkyl groups include theaforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) andcyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include theaforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) andcyclooctyl (C₈). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₄ cycloalkyl. In certain embodiments, thecarbocyclyl includes 0, 1, or 2 C═C double bonds in the carbocyclic ringsystem, as valency permits.

The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more cathocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl. In certain embodiments, the heterocyclyl issubstituted or unsubstituted, 3- to 7-membered, monocyclic heterocyclyl,wherein 1,2, or 3 atoms in the heterocyclic ring system areindependently oxygen, nitrogen, or sulfur, as valency permits.

In some embodiments, a heterocyclyl group is a 5-10 membered nonaromaticring system having ring carbon atoms and 1-4 ring heteroatoms, whereineach heteroatom is independently selected from nitrogen, oxygen, andsulfur (“5-10 membered heterocyclyl”). In some embodiments, aheterocyclyl group is a 5-8 membered non-aromatic ring system havingring carbon atoms and 1-4 rings heteroatoms, wherein each heteroatom isindependently selected from nitrogen, oxygen, and sulfur (“5-8 memberedheterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6membered non-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatom includeazirdinyl, oxiranyl, and thiiranyl. Exemplary 4-membered heterocyclylgroups containing 1 heteroatom include azetidinyl, oxetanyl, andthietanyl. Exemplary 5-membered heterocyclyl groups containing 1heteroatom include tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolindinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining 2 heteroatoms include dioxolanyl, oxathiolanyl anddithiolanyl. Exemplary 5-membered heterocyclyl groups containing 3heteroatoms include triazolinyl, oxadiazolinyl, and thiadiazolinyl.Exemplary 6-membered heterocyclyl groups containing 1 heteroatom includepiperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary6-membered heterocyclyl groups containing 2 heteroatoms includepiperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-memberedheterocyclyl groups containing 3 heteroatoms include triazinyl.Exemplary 7-membered heterocyclyl groups containing 1 heteroatom includeazepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclylgroups containing 1 heteroatom include azocanyl, oxecanyl and thiocanyl.Exemplary bicyclic heterocyclyl groups include indolinyl, isoindolinyl,dihydrobenzofuranyl, dihydrobenzothienyl, tetrahydrobenzothienyl,tetrahydrobenzofuranyl, tetrahydroindolyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, decahydroisoquinolinyl,octahydrochromenyl, octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14 π electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclicor polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system(e.g., having 6, 10, or 14 π electrons shared in a cyclic array) havingring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groupsthat contain one or more nitrogen atoms, the point of attachment can bea carbon or nitrogen atom, as valency permits. Heteroaryl polycyclicring systems can include one or more heteroatoms in one or both rings.“Heteroaryl” includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more carbocyclyl or heterocyclylgroups wherein the point of attachment is on the heteroaryl ring, and insuch instances, the number of ring members continue to designate thenumber of ring members in the heteroaryl ring system. “Heteroaryl” alsoincludes ring systems wherein the heteroaryl ring, as defined above, isfused with one or more aryl groups wherein the point of attachment iseither on the aryl or heteroaryl ring, and in such instances, the numberof ring members designates the number of ring members in the fusedpolycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groupswherein one ring does not contain a heteroatom (e.g., indolyl,quinolinyl, carbazolyl, and the like) the point of attachment can be oneither ring, i.e., either the ring bearing a heteroatom (e.g., 2indolyl)or the rims that does not contain a heteroatom (e.g., 5-indolyl). Incertain embodiments, the heteroaryl is substituted or unsubstituted, 5-or 6-membered, monocyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in theheteroaryl ring system are independently oxygen, nitrogen, or sulfur. Incertain embodiments, the heteroaryl is substituted or unsubstituted, 9-or 10-membered, bicyclic heteroaryl, wherein 1, 2, 3, or 4 atoms in theheteroaryl ring system are independently oxygen, nitrogen, or sulfur.

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom includepyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroarylgroups containing 2 heteroatoms include imidazolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroarylgroups containing 3 heteroatoms include triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include tetrazolyl. Exemplary 6-membered heteroaryl groupscontaining 1 heteroatom include pyridinyl. Exemplary 6-memberedheteroaryl groups containing 2 heteroatoms include pyridazinyl,pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groupscontaining 3 or 4 heteroatoms include triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include azepinyl, oxepinyl, and thiepinyl. Exemplary5,6-bicyclic heteroaryl groups include indolyl, isoindolyl, indazolyl,benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl,benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl,benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl,indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groupsinclude naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include phenanthridinyl, dibenzofuranyl,carbazolyl, acridinyl, phenothiazinyl, phenoxazinyl and phenazinyl.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

A group is optionally substituted unless expressly provided otherwise.The term “optionally substituted” refers to being substituted orunsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl groups are optionally substituted. “Optionallysubstituted” refers to a group which may be substituted or unsubstituted(e.g., “substituted” or “unsubstituted” alkyl, “substituted” or“unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” heteroalkyl, “substituted” or“unsubstituted” heteroalkenyl, “substituted” or “unsubstituted”heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl,“substituted” or “unsubstituted” heterocyclyl, “substituted” or“unsubstituted” aryl or “substituted” or “unsubstituted” heteroarylgroup). In general, the term “substituted” means that at least onehydrogen present on a group is replaced with a permissible substituent,e.g., a substituent which upon substitution results in a stablecompound, e.g., a compound which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, orother reaction. Unless otherwise indicated, a “substituted” group has asubstituent at one or more substitutable positions of the group, andwhen more than one position in any given structure is substituted, thesubstituent is either the same or different at each position. The term“substituted” is contemplated to include substitution with allpermissible substituents of organic compounds, and includes any of thesubstituents described herein that results in the formation of a stablecompound. The present invention contemplates any and all suchcombinations in order to arrive at a stable compound. For purposes ofthis invention, heteroatoms such as nitrogen may have hydrogensubstituents and/or any suitable substituent as described herein whichsatisfy the valencies of the heteroatoms and results in the formation ofa stable moiety. The invention is not intended to be limited in anymanner by the exemplary substituents described herein.

Exemplary carbon atom substituents include halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂, —N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻,—N(OR^(cc))R^(bb), —SH, —SR^(aa), —C(═O)R^(aa), —CO₂H, —CHO,—C(OR^(cc))₂, —CO₂R^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂,—OC(═O)N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—OC(═NR^(bb))R^(aa), —OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—OC(═NR^(bb))N(R^(bb))₂, —NR^(bb)C(═NR^(bb))N(R^(bb))₂,—C(═O)NR^(bb)SO₂R^(aa), —NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa),—SO₂OR^(aa), —OSO₂R^(aa), —S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)₂R^(aa), —OP(═O)₂R^(aa), —P(=O)R^(aa))₂,—OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, —P(═O)₂N(R^(bb))₂,—OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂, —OP(═O)(NR^(bb))₂,—NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂, —P(R^(cc))₂,—P(R^(cc))₃, —OP(R^(cc))₂, —B(R^(aa))₂, —B(OR^(cc))₂, —BR^(aa)(OR^(cc)),C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(dd)groups;

-   -   or two geminal hydrogens on a carbon atom are replaced with the        group ═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa),        ═NNR^(bb)C(═O)OR^(aa), ═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or        ═NOR^(cc);    -   each instance of R^(aa) is, independently, selected from C₁₋₁₀        alkyl, C₁-₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,        heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀        carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14        membered heteroaryl, or two R^(aa) groups are joined to form a        3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,        wherein each alkyl, alkenyl, alkynyl, heteroalkyl,        heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl,        and heteroaryl is independently substituted with 0, 1, 2, 3, 4,        or 5 R^(dd) groups;    -   each instance of R^(bb) is, independently, selected from        hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),        —C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))OR^(aa),        —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),        —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),        —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,        —P(═O)(NR^(cc))₂, C₁₋₁₀ alkyl, C₁₋₁₀ to perhaloalkyl, C₂₋₁₀        alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀alkyl, heteroC₂₋₁₀alkenyl,        heteroC₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered        heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two        R^(bb) groups are joined to form a 3-14 membered heterocyclyl or        5-14 membered heteroaryl ring, wherein each alkyl, alkenyl,        alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,        heterocyclyl, aryl, and heteroaryl is independently substituted        with 0, 1, 2, 3, 4, or 5 R^(dd) groups;    -   each instance of R^(cc) is, independently, selected from        hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀        alkynyl, heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀        alkynyl, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄        aryl, and 5-14 membered heteroaryl, or two R^(cc) groups are        joined to form a 3-14 membered heterocyclyl or 5-14 membered        heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,        heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,        heterocyclyl, aryl, and heteroaryl is independently substituted        with 0, 1, 2, 3, 4, or 5 R^(dd) groups;    -   each instance of R^(dd) is, independently, selected from        halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee),        —ON(R^(ff))₂, —N(R^(ff))₂, —N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff),        —SH, —SR^(ee), —SSR^(ee), —C(═O)R^(ee), —CO₂H, —CO₂R^(ee),        —OC(═O)R^(ee), —OCO₂R^(ee), —C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂,        —NR^(ff)C(═O)R^(ee), —NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂,        —C(═NR^(ff))OR^(ee), —OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee),        —C(═NR^(ff))N(R^(ff))₂, —OC(═NR^(ff))N(R^(ff))₂,        —NR^(ff)C(═NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee),        —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),        —S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,        —C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)₂R^(ee),        —P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆        alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,        heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl, C₃₋₁₀        carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl, 5-10        membered heteroaryl, wherein each alkyl, alkenyl, alkynyl,        heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl,        heterocyclyl, aryl, and heteroaryl is independently substituted        with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two geminal R^(dd)        substituents can be joined to form ═O or ═S;    -   each instance of R^(ee) is, independently, selected from C₁₋₆        alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆        alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl,        C₆₋₁₀ aryl, 3-10 membered heterocyclyl, and 3-10 membered        heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,        heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl,        and heteroaryl is independently substituted with 0, 1, 2, 3, 4,        or 5 R^(gg) groups;    -   each instance of R^(ee) is, independently, selected from        hydrogen, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆        alkynyl, heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆alkynyl,        C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀ aryl and        5-10 membered heteroaryl, or two R^(ff) groups are joined to        form a 3-10 membered heterocyclyl or 5-10 membered heteroaryl        ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,        heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl,        and heteroaryl is independently substituted with 0, 1, 2, 3, 4,        or 5 R^(gg) groups; and    -   each instance of R^(gg) is, independently, halogen, —CN, —NO₂,        —SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆        alkyl)₂, —N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆        alkyl)⁺X⁻, —NH₃ ⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl),        —N(OH)(C₁₋₆alkyl), —NH(OH), —SH, —SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl),        —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl), —OC(═O)(C₁₋₆        alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆ alkyl)₂,        —C(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆        alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆        alkyl)₂, —NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆        alkyl), —OC(═NH)(C₁₋₆ alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆        alkyl)₂, —C(═NH)NH(C₁₋₆ alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆        alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl), —OC(NH)NH₂, —NHC(NH)N(C₁₋₆        alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl), —SO₂N(C₁₋₆ alkyl)₂,        —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂C₁₋₆ alkyl,        —OSO₂C₁₋₆ alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃, —OSi(C₁₋₆        alkyl)₃—C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,        —C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl,        —P(═O)₂(C₁₋₆ alkyl), —P(═O)(C₁₋₆ alkyl)₂, —PP(═O)(C₁₋₆ alkyl)₂,        —OP(═O)(OC₁₋₆ alkyl)₂, C₁₋₆ alkyl, perhaloalkyl, C₂₋₆ alkenyl,        C₂₋₆ alkynyl, heteroC₁₋₆alkyl, heteroC₂₋₆alkenyl,        heteroC₂₋₆alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered        heterocyclyl, 5-10 membered heteroaryl; or two geminal R^(gg)        substituents can be joined to form ═O or ═S; wherein X⁻ is a        counterion.

In certain embodiments, the carbon atom substituents are independentlyhalogen, substituted (e.g., substituted with one or more halogen) orunsubstituted C₁₋₆ alkyl, —OR^(aa), —SR^(aa), —N(R^(bb))₂, —CN, —SCN,—NO₂, —C(═O)R_(aa), —CO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)R^(aa),—OCO₂R^(aa), —OC(═O)N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),or —NR^(bb)C(═O)N(R^(bb))₂. In certain embodiments, the carbon atomsubstituents are independently halogen, substituted (e.g., substitutedwith one or more halogen) or unsubstituted C₁₋₆ alkyl, —OR^(aa),—SR^(aa), —N(R^(bb))₂, —CN, —SCN, —NO₂, —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), or —NR^(bb)C(═O)N(R^(bb))₂,wherein R^(aa) is hydrogen, substituted (e.g., substituted with one ormore halogen) or unsubstituted C₁₋₆ alkyl, an oxygen protecting group(e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM, THP, t-Bu, Bn, allyl,acetyl, pivaloyl, or benzoyl) when attached to an oxygen atom, or asulfur protecting group (e.g., acetamidomethyl, t-Bu, 3-nitro-2-pyridinesulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl) when attached to asulfur atom; and each R^(bb) is independently hydrogen, substituted(e.g., substituted with one or more halogen) or unsubstituted C₁₋₆alkyl, or a nitrogen protecting group (e.g., Bn, Boc, Cbz, Fmoc,trifluoroacetyl, triphenylmethyl, acetyl, or Ts). In certainembodiments, the carbon atom substituents are independently halogen,substituted (e.g., substituted with one or more halogen) orunsubstituted C₁₋₆ alkyl, —OR^(aa), —N(R^(bb))₂, —CN, —SCN, or —NO₂. Incertain embodiments, the carbon atom substituents are independentlyhalogen, substituted (e.g., substituted with one or more halogenmoieties) or unsubstituted C₁₋₆ alkyl, —OR^(aa), —SR^(aa), —N(R^(bb))₂,—CN, —SCN, or —NO₂, wherein R^(aa) is hydrogen, substituted (e.g.,substituted with one or more halogen) or unsubstituted C₁₋₆ alkyl, anoxygen protecting group (e.g., silyl, TBDPS, TBDMS, TIPS, TES, TMS, MOM,THP, t-Bu, Bn, allyl, acetyl, pivaloyl, or benzoyl) when attached to anoxygen atom, or a sulfur protecting group (e.g., acetamidomethyl, t-Bu,3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl, or triphenylmethyl)when attached to a sulfur atom; and each R^(bb) is independentlyhydrogen, substituted (e.g., substituted with one or more halogen) orunsubstituted C₁₋₆ alkyl, or a nitrogen protecting group (e.g., Bn, Boc,Cbz, Fmoc, trifluoroacetyl, triphenylmethyl, acetyl, or Ts).

In certain embodiments, the molecular weight of a carbon atomsubstituent is lower than 250, lower than 200, lower than 150, lowerthan 100, or lower than 50 g/mol. In certain embodiments, a carbon atomsubstituent consists of carbon, hydrogen, fluorine, chlorine, bromine,iodine, oxygen, sulfur, nitrogen, and/or silicon atoms. In certainembodiments, a carbon atom substituent consists of carbon, hydrogen,fluorine, chlorine, bromine, iodine, oxygen, sulfur, and/or nitrogenatoms. In certain embodiments, a carbon atom substituent consists ofcarbon, hydrogen, fluorine, chlorine, bromine, and/or iodine atoms. Incertain embodiments, a carbon atom substituent consists of carbon,hydrogen, fluorine, and/or chlorine atoms.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine(chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “oxo” refers to the moiety ═O.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a positively charged group in order to maintainelectronic neutrality. An anionic counterion may be monovalent (i.e.,including one formal negative charge). An anionic counterion may also bemultivalent (i.e., including more than one formal negative charge), suchas divalent or trivalent. Exemplary counterions include halide ions(e.g., F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HCO₃ ⁻, HSO₄ ⁻,sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate,p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate,naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate,ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions(e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, gluconate, and the like), BF₄ ⁻, PF₄ ⁻, PF₆ ⁻, AsF₆ ⁻, SbF₆⁻, B[3,5-(CF₃)₂C₆H₃]₄]⁻, B(C₆F₅)₄ ⁻, BPh₄ ⁻, Al(OC(CF₃)₃)₄ ⁻, andcarborane anions (e.g., CB₁₁H₁₂ ⁻ or (HCB₁₁Me₅Br₆)⁻). Exemplarycounterions which may be multivalent include CO₃ ²⁻, HPO₄ ²⁻, PO₄ ³⁻,B₄O₇ ²⁻, SO₄ ²⁻, S₂O₃ ²⁻, carboxylate anions (e.g., tartrate, citrate,fumarate, maleate, malate, malonate, gluconate, succinate, glutarate,adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates,aspartate, glutamate, and the like), and carboranes.

EXAMPLES Example 1 Sensing Intracellular Calcium Ions Using aManganese-Based MRI Contrast Agent Building Block for Intracellular MRICalcium Sensors

To examine the potential of Mn-PDA contrast agents to function asbuilding blocks for the sensor design of FIG. 1A, MRI measurements wereperformed to assess the potential of BAPTA to interact with threeparamagnetic chelates (FIG. 1A, inset). Addition of 1.1 equivalents ofBAPTA to two of the Mn-PDA complexes, MnL1 and MnL3, producedsignificant effects on the T₁-weighted MRI contrast induced by thesecompounds in buffer. These BAPTA-dependent contrast changes werereversed by addition of equimolar Ca²⁺ (FIG. 1B). This was consistentwith Mn-PDA moieties competing with Ca²⁺ for binding to BAPTA molecules.The changes were quantified in terms of T₁ relaxivity (r₁) values, whichwere defined as the slope of the T₁ relaxation rate (1/T₁=R₁) vs.concentration of each paramagnetic complex. Calcium-dependent changes inr₁ observed for MnL1 and MnL3 in the presence of BAPTA were 105% and23%, respectively; both were significant.

Mixtures of MnL1 and MnL3 with BAPTA both showed calcium-dependent MRIproperties. The spectroscopic behavior of MnL1 and MnL3 in the presenceof BAPTA over time was examined, and it was found that theMnL1-containing mixture displayed sharp changes indicative ofdegradation, while the MnL3 mixture remained unperturbed and apparentlystable (FIG. 1D). These results were confirmed by high resolution massspectrometry of the two mixtures. Even after 24 hours, the MnL3 mixturewith BAPTA displayed a prominent base peak associated with [MnL3+Cl]⁻(m/z=419.77) but no peak for demetallated L3 (FIG. 1D). In contrast, theMnL1 mixture with BAPTA developed a strong peak for uncomplexed L1(m/z=347.37), indicating dissociation of the MnL1 complex. These resultsindicated that MnL3 and BAPTA constituted a suitable pair of buildingblocks for intracellular calcium sensor construction.

Synthesis and Characterization of ManICS1 and ManICS1-AM

To form a first manganese-based intracellular calcium sensor (ManICS1)from MnL3 and BAPTA, ManICS1 and its AM ester derivative ManICS1-AM weresynthesized using a series of multistep reactions starting from5-methyl-2-nitrophenol (1) (FIG. 6). Synthesis of compound 7 as theparent BAPTA derivative for preparing ManICS was adapted from theprocedure reported. by Grynkiewicz et al. (Grynkiewicz G; Poenie M; andTsien R Y. J. Biolog. Chem. 1985, 260, 3440-3450). Conversion of thealdehyde group of this compound to a carboxylic acid was performedthrough Pinick oxidation, which resulted in high yields without usingtransition metal catalysis. Successive attachment of a polyethyleneglycol linker and carboxylate-modified L3 derivative to compound 7 wereachieved using benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate, which produced higher yields than other couplingreagents. The resulting compound 11 was metallated to form ManICS1-AM byreaction with Mn(OAc)₃.2(H₂O) in acetonitrile in the presence ofN,N-diisopropylethylamine. Enzymatic hydrolysis of ManICS1-AM or directcoupling of compound 12 to MnL3COOH afforded ManICS1 in moderate yields(FIG. 6).

The calcium responsiveness of ManICS 1 was measured in buffer bytitrating the compound with calcium chloride over a concentration rangerelevant to intracellular signaling. Images indicated clear enhancementof T₁-weighted MRI intensity as calcium ions were added. (FIG. 3A). Noimage changes were observed when Mg²⁺ was added in place of Ca²⁺,indicating specificity of the responses to calcium. Apparent relaxivityvalues were determined for each condition (FIG. 3B). The data indicatedthat calcium binding induced a 34% increase in relaxivity of ManICS1,with r₁ values ranging from 3.8 mM⁻¹s⁻¹ to 5.1 mM⁻¹s⁻¹ over the full.calcium range. As expected, no significant r₁ change was observed withMg²⁺ titration. The dissociation constant for calcium binding to ManICS1was determined to be 18 micromolar (μM). This value is higher than mosthigh affinity fluorescent indicators used for intracellular Ca²⁺imaging, but is still sufficient for transducing [Ca²⁺] fluctuations byas little as 1 micromolar (μM) from baseline into T₁-weighted imagechanges of ˜1% or higher, equivalent to signals commonly detected infunctional MRI experiments.

To probe the mechanism of calcium sensing by ManICS1, a series ofexperiments were performed. The apparent calcium affinity of the probewas relatively low for the BAPTA derivative that was used, which in thecontext of fluorescent sensors generally confers calcium binding withsubmicromolar calcium K_(d) values. To further evaluate calcium sensing,a manganese-free ManICS1 analog was formed and its calcium affinity wasmeasured by titration with a spectroscopic readout. A separate calciumtitration series was also performed with three different concentrationsof ManICS1. Data from the three datasets produced K_(d) estimates thatdid not differ significantly from one another.

To demonstrate that the ester groups of ManICS1-AM are capable ofundergoing cleavage in the cytosolic milieu, the compound was incubatedin clarified cell lysate (10% by volume) and the high performance liquidchromatography (HPLC) time course was compared to that of ManICS1 andManICS1-AM incubated in buffer alone. It was found that five hours ofexposure to cell lysate was sufficient to result in complete conversionof the ManICS1-AM HPLC peak into a product that eluted at the same timeas ManICS1 (FIG. 3C). The identity of this product as ManICS1 wasconfirmed by mass spectrometry, indicating that all four esters ofManICS1-AM were released within the incubation time. As a further testof the behavior of ManICS1 ester derivatives, ManICS1-Et wassynthesized, in which all four BAPTA carboxylates were modified by ethylesters rather than AM groups. HPLC data indicated that ManICS1-Etincubation did not yield ManICS1 after five hour incubation with lysate.Thus, in some embodiments, genetically-targeted cleavage may be usefulto bring about intracellular accumulation of ManICS-Et, in whichappropriately selective esterases are expressed ectopically.

ManICS1-AM Labeled Cells and Enabled Intracellular Calcium-Sensitive MRI

To validate the potential for ManICS1-AM to enable readouts ofintracellular calcium by T₁-weighted MRI, the ability of ManICS1-AM tolabel cells and subsequently be retained was first examined. CulturedHEK293 cells were incubated with ManICS1-AM or ManICS1 followed bywashing and then MRI analysis. It was found that cells incubated withthe two agents underwent a comparable increase in R, compared withcontrol cells (FIG. 4A), suggesting that both agents were internalizedto some extent. When a delay was inserted, followed by additionalwashing, between the labeling period and the MRI, it was found thatManICS1-treated cells returned quickly to baseline values within 5hours, while cells treated with ManICS1-AM maintained elevated contrastfor up to 24 hours. To assess the subcellular localization of manganesefollowing ManICS1-AM or ManICS labeling, cells were fractionated intocytosolic, organellar, and membranous compartments and the samples wereanalyzed by inductively coupled plasma optical emission spectroscopy(ICP-OES). The ICP-OES results indicated that ManICS1-AM labelingproduced long-lasting cytosolic elevations in manganese content, whileManICS labeling produced shorter-lived organellar accumulation. Theseresults were consistent with the hypothesis that ManICS1-AM penetratescells and undergoes cytosolic accumulation as diagrammed in FIG. 1A,while ManICS1 in its acidic form may be internalized to some extent viaendocytosis.

To assess the potential for internalized ManICS1 to transduce cytosoliccalcium concentrations into R₁ changes detectable by MRI, culturedHEK293 cells were labeled again with ManICS1-AM and then challenged withstimuli known to elevate intracellular [Ca²⁺]. Cells treated withthapsigargin and carbachol did not show elevated R₁, whereas addition ofcalcimycin or arachidonic acid produced substantial increases in meanR₁. The same four stimulants were applied separately to cells prelabeledwith the fluorescent calcium indicator derivative Fura-2-AM.Fluorescence ratio measurements from these cells closely paralleled theresults obtained by MRI analysis of ManICS1-AM-labeled cells, indicatingthat the variable MRI results were indicative of actual intracellularcalcium responses.

As a further test of the calcium-dependent contrast properties conferredby ManICS1-AM, intracellular calcium levels were titrated and theprofile of resulting R₁ changes was examined. Intracellular calciumlevels were regulated by controlling extracellular calcium using astandard buffering system in the presence of the ionophore calcimycin,which equilibrates calcium levels across the cell membrane. Resultsdemonstrated progressive changes in R₁ as [Ca²⁺] ranged from 1micromolar (μM) to 100 micromolar (μM), establishing a midpoint (EC₅₀)for intracellular calcium sensing by ManICS1 of about 5 micromolar (μM)(FIG. 4D). As a further experiment, cells in high calcium conditionswere treated, washed, and then returned to low calcium conditions beforeMRI was performed. Cells treated in this way displayed the lowerT₁-weighted MRI signal characteristic of low [Ca²⁺] conditions,indicating that elevated calcium did not produce irreversible increasesin

These results showed MRI-based measurement of intracellular calciumusing a manganese-based MRI contrast agent that parallelled propertiesof fluorescent probes for cytosolic Ca²⁺ imaging. In this work, ManICS1was synthesized, which incorporated membrane-permeable building blocksand involved the AM ester-based approach for cell labeling andintracellular trapping of the probe. ManICS1 reported calcium levelsanalogously to the widely used optical calcium sensor Fura-2. The novelprobe may be used for MRI-based spatiotemporal mapping of calciumsignaling processes in contexts where optical imaging approaches havepreviously been successful.

Example 2 Methods and Materials Magnetic Resonance Imaging

Most MRI data were acquired on a 7 T Bruker Biospec system using aT₁-weighted 2D gradient echo sequence (echo time, TE=5 ms, repetitiontime, TR=100 ms; flip angle, FA=65 degrees (°)). Longitudinal relaxivity(r₁) measurements were acquired using a 2D spin echo sequence (TE=11 ms,TR=125, 200, 400, 800, 1500, 3000, and 5000 ms), with in-planeresolution of 200×200 microns squared ((μm)²) and 2 mm slice thickness.R₁ maps and values were generated using an MRI Analysis CalculatorPlugin for ImageJ (National Institutes of Health, Bethesda, Md.) orMatlab scripts (Mathworks, Natick, Mass.). Stimulus-dependent R₁ changesin cells were calculated as ΔR₁/R₁=[R₁(incubated cells)−R₁(naivecells)]/R₁(naive cells). Statistical comparisons between pairedconditions were performed using Student's t-test, and all error barsdenote the standard error of the mean from multiple measurements, unlessotherwise noted.

In Vitro Characterization of ManICS1 and ManICS1-AM

ManICS1, calcium, and magnesium stock solutions were all prepared in 25mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.4, with 100 mM KCl.Titration series were prepared as separate triplicates for each datapoint in the presence of constant concentrations of ManICS1, confirmedsubsequently by ICP-OES analysis. Samples were arrayed into microtiterplates and measured by MRI using a Bruker (Billerica, Mass.) Avance 7 Tscanner. Unused wells were filled with buffer, and imaging was performedon a 2 mm slice through the sample. Calcium affinity of manganese-freeManICS1 was determined using optical spectroscopy.

Cell Labeling with ManICS1-AM

HEK293 cells (Freestyle 293-F, Thermo Fischer Scientific, Waltham,Mass.) were cultured and prepared for relaxometry. To assess uptake,cells were exposed to contrast agents in media for 30 min, washed withHank's buffered saline solution (HBSS), and then immediately pelleted bycentrifugation at 750 g for imaging, incubated in media again forvarying time intervals, or stimulated with pharmacological agents.Subcellular fractionation analysis of ManICS1-AM-labeled cells wasconducted. Cells were incubated with agent for 30 min, washed in HBSSand permeablized on ice with saponin to collect cytosolic fractions foranalysis with ICP-OES.

Measurement of Intracellular Calcium Responses

For stimulation experiments, cells were incubated with 10 micromolar(μM) ManICS1-AM for 2 h for labeling and AM ester cleavage.Pharmacological stimulation was conducted by adding 10 micromolar (μM)thapsigargin, 5 micromolar (μM) charbocol, 5 micromolar (μM) calcimycin,or 10 micromolar (μM) arachidonic acid (Sigma-Aldrich, St. Louis, Mo.).To test reversibility of the calcium response, cells were prepared withManICS1-AM and 5 micromolar (μM) calcimycin maintained for the durationof the experiment, washed in calcium free media, divided into twoaliquots to be washed again or returned to 2 mM Ca²⁺. For the titrationcurve cells were maintained in 5 micromolar (μM) calcimycin first in100× volume calcium-free buffer, then while incubated in 100× volumemedia with 0-1 mM calcium for 1.5 min at 37 degrees Celsius (° C.).

For comparison stimulation measurements performed with Fura-2, adherentHEK293 cells were seeded onto a 96 well plate at 5,000 cells/well andgrown for two days until 90% confluent. Cells were then incubated for 45min in 10 micromolar (μM) Fura-2 and then washed with media. Stimulants(10 micromolar (μM) thapsigargin, 5 micromolar charbocol, 5 micromolar(μM) calcimycin, or 10 micromolar (μM) arachidonic acid) were quicklyadded to multiple wells via multipipettor and fluorescence output wasmeasured using a plate reader. Measurements were repeated every 5minutes for 40 minutes to chart the time course of calcium concentrationchanges at room temperature.

Example 3 ManICS1-AM Labels Cells and Enables IntracellularCalcium-Sensitive MRI

To test the ability of ManICS1-AM to enable readouts of intracellularcalcium by T₁-weighted MRI, its propensity was first examined toaccumulate within cells. Cultured HEK293 cells were incubated with 10 μMManICS1-AM or ManICS1 for 30 minutes, followed by washing and MRIanalysis (FIG. 7A). It was found that cells incubated with the cellpermeable ManICS1-AM underwent a substantial increase in R₁ thatpersisted above basal levels for up to 24 h, while cells labeled withManICS1 experienced a somewhat lesser increase in R₁ that returned tobaseline within 5 h. To assess the subcellular localization of manganesefollowing ManICS1-AM or ManICS1 labeling, cytosolic fractions wereisolated from cell lysates and the samples were analyzed by inductivelycoupled plasma mass spectrometry (ICP-MS). The ICP-MS results indicatedthat only ManICS1-AM labeling produced cytosolic elevations in manganesecontent (FIG. 7A inset). These results are consistent with thehypothesis that ManICS1-AM penetrates cells and is retained in thecytosol (FIG. 1A).

To assess the potential for internalized ManICS1 to reportstimulus-induced cytosolic calcium concentrations as changes detectableby MRI, HEK293 cells were again labeled with ManICS1-AM; then the cellswere challenged with pharmacological agents that elevate cytosoliccalcium levels (FIG. 7C). Calcimycin and arachidonic acid both produced8-10% increases in mean R₁ values recorded within 20 min of stimulation.Cells treated with thapsigargin or carbachol, which are thought to causeonly short-lived Ca²⁺ responses, did not show an elevated R₁. Controlmeasurements using the fluorescent calcium indicator derivativeFura-2FF-AM collected using similar stimulus conditions closelyparalleled the results obtained by MRI of ManICS1-AM-labeled cells (FIG.7D), showing that the MRI results provide an accurate measure ofintracellular calcium perturbations.

To examine the reversibility and dynamics of ManICS1-based calciumsensing the optogenetic Ca²⁺ actuator BACCS2 was used to stimulatecontrast agent-labeled cells while performing functional imaging withMRI. In preparation for these experiments, cells were embedded in a gelmatrix that permitted effective Ca²⁺ exchange at high cell density.BACCS2-expressing cells loaded with ManICS1-AM showed dynamiclight-dependent image changes averaging 0.8±0.2% in amplitude (FIG. 7E),matching a time course obtained using optical measurements of cellsloaded with the fluorescent calcium indicator X-Rhod-1 (FIG. 7F).Stimulation of ManICS1-AM-labeled cells that did not express BACCS2 orMnL3-labeled cells expressing BACCS2 produced negligible MRI effects.Both were significantly lower than those observed with ManICS1-AM inBACCS2-expressing cells (t-test p≤10-5), revealing the calciumspecificity of T₁-weighted imaging signals obtained with ManICS1-AMinside living cells.

Example 4 ManICS1-AM Permits Detection of Deep Brain Activation in Rats

To determine whether ManICS1-AM could enable detection of intracellularcalcium signals in vivo, the probe was injected into the brains of adultrats and responses were examined to stimulation with potassium ions,which induce neural depolarization and calcium concentration changes inbrain tissue. Intracranial infusion of ManICS1-AM into the striatumresulted in substantial T₁-weighted MRI signal enhancement over a 4 mmdiameter region around the injection site, as well as more remote tissuealong the ventricles (FIGS. 8A and 8B). The contrast enhancementpersisted for over 90 min without significant loss of signal (t-testp=0.42, n=4) (FIG. 8C), differing markedly from the behavior ofhydrophilic MRI contrast agents that do not enter cells and thattypically clear from the rodent brain within two hours.

Infusion of artificial cerebrospinal fluid (aCSF) formulatedisotonically with 125 mM KCl elicited a robust signal change proximal tothe infusion site in ManICS1-AM-infused brain areas (FIG. 8D). TheManICS1-dependent signal rose quickly, with an average signal change of5.8±1.2% at stimulus offset that subsided slowly to baseline after theKCl infusion stopped (FIG. 8E). KCl injection in the presence of thecalcium-insensitive contrast agent MnL1F—a close variant of the solublecell-permeable MnL1 chelate—or infusion of standard aCSF containing 125mM NaCl in the presence of ManICS1-AM elicited negligible mean responsesof −0.2±0.8% and 1.2±1.2%, respectively, at stimulus offset with respectto baseline. The signal change observed under the ManICS1-AM K⁺stimulation condition differed significantly from signals observed underboth control conditions (t-test p≤0.016, n=5) (FIG. 8F), consistent withthe expected calcium-sensing mechanism of ManICS1 and with resultsobtained in cells.

These experiments thus demonstrate a cell--permeable manganese-based MRIcontrast agent that emulates properties of fluorescent probes forcytosolic Ca²⁺ imaging, and that can detect signaling events in deeptissue. The ManICS1 calcium sensor introduced here incorporatesmembrane-permeable building blocks and can exploit the AM ester-basedapproach for cell labeling and cytosolic trapping of the probe. Uponinternalization, ManICS1-AM reports calcium levels consistent withreadouts from optical calcium sensors and compatible with T₁-weightedfunctional MRI in rat brain, suggesting that the new probe could be usedfor spatiotemporal mapping of calcium signaling processes previouslyaccessible only to optical imaging approaches, but with the expandeddepth and field of view afforded by MRI.

While several embodiments of the present invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and/or structures for performing thefunctions and/or obtaining the results and/or one or more of theadvantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto, the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, kit, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,kits, and/or methods, if such features, systems, articles, materials,kits, and/or methods are not mutually inconsistent, is included withinthe scope of the present invention.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified unless clearly indicated to the contrary. Thus,as a non-limiting example, a reference to “A and/or B,” when used inconjunction with open-ended language such as “comprising” can refer, inone embodiment, to A without B (optionally including elements other thanB); in another embodiment, to B without A (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” and the like are to be understoodto be open-ended, i.e., to mean including but not limited to. Only thetransitional phrases “consisting of” and “consisting essentially of”shall be closed or semi-closed transitional phrases, respectively, asset forth in the United States Patent Office Manual of Patent ExaminingProcedures, Section 2111.03.

1. A sensor comprising the structure, Y-L-Z, wherein: Y is an analytebinding moiety; Z is a lipophilic, branched chelating moiety; and L is alinker that covalently links Y and Z.
 2. The sensor of claim 1, whereinthe sensor further comprises an additional Y, Z, L, L-Y, or L-Zcovalently bound to Y or Z.
 3. The sensor of claim 1, wherein thelipophilic, branched chelating moiety is bound to a paramagnetic metalion.
 4. (canceled)
 5. The sensor of claim 1, wherein the analyte is acell signaling ion.
 6. The sensor of claim 5, wherein the cell signalingion is a cation selected from the group consisting of: Calcium²⁺,Sodium⁺, Potassium⁺, Magnesium²⁺, and Zinc²⁺. 7-9. (canceled)
 10. Thesensor of claim 1, wherein the analyte binding moiety contains at leastone aromatic group.
 11. The sensor of claim 1, wherein the analytebinding moiety contains at least one aromatic group selected from thegroup consisting of monocyclic aryl, bicyclic aryl, monocyclicheteroaryl, and bicyclic heteroaryl.
 12. The sensor of claim 1, whereinthe lipophilic, branched chelating moiety has a planar configurationwhen chelating a paramagnetic metal ion. 13-14. (canceled)
 15. Thesensor of claim 1, wherein the lipophilic, branched chelating moietycomprises an aromatic group.
 16. The sensor of claim 1, wherein thelipophilic, branched chelating moiety comprises one or more cyclicmoieties.
 17. The sensor of claim 16, wherein each of the cyclicmoieties is independently selected from the group consisting ofmonocyclic aryl, bicyclic aryl, monocyclic heteroaryl, and bicyclicheteroaryl.
 18. (canceled)
 19. The sensor of claim 18, wherein each ofthe three cyclic moieties is independently phenyl, pyridinyl, anilinylor pyrrolyl.
 20. The sensor of claim 1, wherein the lipophilic, branchedchelating moiety is of the formula:

wherein: M is metal ion, optionally a paramagnetic metal ion; X is N orC—OH; each instance of R^(A) is independently halogen, unsubstitutedC₁₋₆ alkyl, C₁₋₆ alkyl substituted with one or more instances ofhalogen, oxo, or —OR^(a); each instance of R^(a) is independentlyunsubstituted C₁₋₆ alkyl or C₁₋₆ alkyl substituted with one or moreinstances of halogen; k is 0, 1, 2, or 3; each instance of R^(B) isdirectly attached to any one of the 3a- to 6a-positions and isindependently halogen, unsubstituted C₁₋₆ alkyl, C₁₋₆ alkyl substitutedwith one or more instances of halogen, oxo, or —OR^(a), or two instancesof R^(B) are joined to form a phenyl ring, wherein the phenyl ringformed by joining two instances of R^(B) is unsubstituted or substitutedwith 1, 2, 3, or 4 instances of substituents independently selected fromthe group consisting of halogen, —OR^(a), unsubstituted C₁₋₆ alkyl, andC₁₋₆ alkyl substituted with one or more instances of halogen; m is 0, 1,2, 3, or 4; each instance of R^(C) is independently halogen,unsubstituted C₁₋₆ alkyl, C₁₋₆ alkyl substituted with one or moreinstances of halogen, oxo, or —OR^(a), or two instances of R^(C) arejoined to form a phenyl ring, wherein the phenyl ring formed by joiningtwo instances of R^(C) is unsubstituted or substituted with 1, 2, 3, or4 instances of substituents independently selected from the groupconsisting of halogen, —OR^(a), unsubstituted C₁₋₆ alkyl, and C₁₋₆ alkylsubstituted with one or more instances of halogen; and n is 0, 1, 2, 3,or
 4. 21-26. (canceled)
 27. The sensor of claim 1, wherein the analytebinding moiety comprises a selective calcium chelating moiety derivedfrom 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA).28-29. (canceled)
 30. The sensor of claim 27, wherein the selectivecalcium chelating moiety derived from BAPTA does not comprise themoieties —C(═O)OH.
 31. The sensor of claim 20, wherein the analytebinding moiety is of the formula:

wherein: L¹ is unbranched C₂₋₈ alkylene, wherein: 0, 1, 2, or 3methylene units of the unbranched C₂₋₈ alkylene are independentlyreplaced with O or N(R^(F)), as valency permits, wherein each instanceof R^(F) is independently H or unsubstituted C₁₋₆ alkyl; and eachmethylene unit of the unbranched C₂₋₈ alkylene is independentlyunsubstituted or substituted with 1 or 2 instances of substituentsindependently selected from the group consisting of oxo, —OR^(a),halogen, unsubstituted C₁₋₆ alkyl, and C₁₋₆ alkyl substituted with oneor more instances of halogen; each instance of R^(a) is independentlyunsubstituted C₁₋₆ alkyl or C₁₋₆ alkyl substituted with one or moreinstances of halogen; each instance of R^(D) is independently halogen,unsubstituted C₁₋₆ alkyl, C₁₋₆ alkyl substituted with one or moreinstances of halogen, or —OR^(a); p is 0, 1, 2, or 3; each instance ofR^(E) is independently halogen, unsubstituted C₁₋₆ alkyl, C₁₋₆ alkylsubstituted with one or more instances of halogen, or —OR^(a); q is 0,1, 2, 3, or 4; each instance of r is independently 0, 1, 2, or 3; eachinstance of R is independently H or substituted or unsubstituted C₁₋₆alkyl. 32-43. (canceled)
 44. The sensor of claim 20, wherein the analytebinding moiety is a polyaminocarboxylate chelator.
 45. The sensor ofclaim 44, wherein the polyaminocarboxylate chelator is selected from thegroup consisting of ethylenediaminetetraacetic acid (EDTA), ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA),diethylenetriaminepentaacetic acid (DTPA),2-[(2-amino-5-methylphenoxy)methyl]-6-methoxy-8-aminoquinoline-N,N,N′,N′-tetraaceticacid (quin-2),1-(2-nitro-4,5-dimethoxyphenyl)-1,2-diaminoethane-N,N,N′,N′-tetraaceticacid (DM-nitrophen), and o-aminophenol-N,N,O-triacetic acid (APTRA).46-47. (canceled)
 48. The sensor of claim 20, wherein the analytebinding moiety is a metal ion-selective crown ether.
 49. (canceled) 50.The sensor of claim 1, wherein the linker, L, is an unbranched C₄₋₄₀alkylene, unbranched C₄₋₄₀ alkenylene, or unbranched C₄₋₄₀ alkynylene,wherein: 0, 1, or more methylene units of the unbranched C₄₋₄₀ alkylene,unbranched C₄₋₄₀ alkenylene, or unbranched C₄₋₄₀ alkynylene areindependently replaced with O, N, N(R^(F)), C(═O)O, C(═O)NR^(F), S,optionally substituted carbocyclyl, optionally substituted heterocyclyl,optionally substituted aryl, or optionally substituted heteroaryl asvalency permits, wherein each instance of R^(F) is independently H orunsubstituted C₁₋₆ alkyl; each methylene unit of the unbranched C₄₋₄₀alkylene, unbranched C₄₋₄₀ alkenylene, or unbranched C₄₋₄₀ alkynylene isindependently unsubstituted or substituted with 1 or 2 instances, asvalency permits, of substituents independently selected from the groupconsisting of oxo, —OR^(a), halogen, unsubstituted C₁₋₆ alkyl, and C₁₋₆alkyl substituted with one or more instances of halogen; and eachinstance of R^(a) is independently unsubstituted C₁₋₆ alkyl or C₁₋₆alkyl substituted with one or more instances of halogen. 51-53.(canceled)